DNA sequences coding for a polyol carrier and their use, in particular for the preparation of transgenic plants

ABSTRACT

The invention concerns the use of a DNA sequence coding for a polyol carrier, in plants and fungi, such as polyols having a main chain containing 5 to 8 carbon atoms, in particular 5 to 7 carbon atoms, more preferably 6 carbon atoms, the polyols being advantageously selected among mannitol, sorbitol, dulcitol, galactitol, inositol, myo-inositol, ribitol and xylitol, and being preferably mannitol, for preparing transgenic plants.

The invention relates to DNA sequences coding for a polyol carrier andtheir use, in particular for the preparation of transgenic plants.

The plants are capable of synthesizing, via photosynthesis, primarycompounds such as glucides by using light energy. Only certain organs ofthe plant, mainly the adult leaves, are capable of manufacturing andexporting the glucides towards the storage organs, such as the tubers,the seeds and the fruits, used in human and animal foodstuffs.

In the majority of plants, the main glucide transported is saccharose,but in a large number of plants, other compounds are also transportedsuch as polyols of which mannitol is an example.

Polyols are, like saccharose, primary products of photosynthesis. It hasfurthermore been estimated that approximately 30% of the globalproduction of primary carbon was used for the synthesis of polyols.

Polyols, cyclic or non-cyclic, are very widespread in plants; they arelow-molecular weight, very soluble and non-reducing compounds. The threenon-cyclic polyols (alditols) which are most widespread amongst theAngiosperms are galactitol, sorbitol and mannitol. Sorbitol is the mainphotosynthetic product in several species of Rosaceae such as the apple,the pear, the peach and the plum.

Mannitol, the most widespread of the alditols, is present in more than100 species of higher plants, in particular in the Rubiaceae (coffee),the Oleaceae (privet, ash, olive) and the Apiaceae (celery, carrot,parsley) (Lewis, 1984). It is produced in the mesophyll cells (cellscontaining chlorophyll). To circulate, it must re-enter the sieve tubes(veins). However, there is no continuity between the mesophyll cells andthe sieve tubes: a mannitol carrier is therefore needed. In this way,the mannitol leaves the mesophyll cells and uses the carrier to enterthe sieve tubes.

The compounds synthesized in the adult leaves are transported towardsthe storage organs and cross a certain number of membranes using thespecialized proteins that are the carriers. These carriers play aconsiderable role in the plant as they are essential for its growth.

The existence of a mannitol carrier in a plant such as celery has beenshown by different biochemical experiments (Salmon et al., 1995). Thispublication has shown that there was a mannitol carrier in celery andthat the expression of this carrier was very sizeable in the tissues ofthe phloem. However, nothing is said as to the identification of themannitol carrier.

If numerous carriers of sugars, such as saccharose and the hexoses havebeen cloned during the course of the last few years, none of them iscapable of transporting polyol.

At present, no carrier of linear polyol has been identified in a livingorganism. In bacteria, a multienzymatic system capable of bothtransporting and phosphorylating mannitol has been described (Boer etal., 1994). However, such systems have never been described in thehigher organisms.

A subject of the invention is carriers of polyols in plants and fungi,and their DNA sequences.

A subject of the invention is also the use of DNA sequences of a polyolcarrier for obtaining transgenic plants.

A subject of the invention is also the use of DNA sequences of a polyolcarrier, in particular within the scope of obtaining plants resistant topathogens or plants resistant to saline stress.

A subject of the invention is also the use of DNA sequences of a polyolcarrier within the scope of a method of screening genetically modifiedplants.

The invention relates to the use of a DNA sequence coding for a linearpolyol carrier, in plants and fungi,

such as polyols having a main chain containing 5 to 8 carbon atoms, inparticular 5 to 7 carbon atoms, in particular 6 carbon atoms, thesepolyols being advantageously chosen from mannitol, sorbitol, dulcitol,galactitol, inositol, ribitol and xylitol, and being in particularmannitol, for the preparation of transgenic plants.

In the expression “plants and fungi”, are included algae, mosses(Bryophytes), ferns (Pteridophytes), higher plants (Gymnosperms andAngiosperms) and fungi.

It can be recalled that, by definition, a polyol is a “polyalcohol”containing as many alcohol functions as carbon atoms. It can also bespecified that the terms polyol, polyalcohol and alcohol sugar areequivalents.

According to an advantageous embodiment, the invention relates to theuse, for the preparation of transgenic plants, of a DNA sequence chosenfrom one of the following sequences: SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9 and SEQ ID NO: 10.

SEQ ID NO: 1 is a new nucleic acid sequence identified in celery (Apiumgraveolens L.), coding for a mannitol carrier.

SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 are sequences of nucleicacids coding for proteins, the functions of which were unknown untilnow.

SEQ ID NO: 3 (Beet 1) and SEQ ID NO: 4 (Beet 2) originate from theBeetroot (Beta vulgaris).

SEQ ID NO: 5 (Pst 1), SEQ ID NO: 6 (Pst 2), SEQ ID NO: 7 (Pst 3), SEQ IDNO: 8 (Pst 4) and SEQ ID NO: 9 (Pst 5) originate from Arabidopsisthaliana.

SEQ ID NO: 10 (Bs) originates from Bacillus subtilis.

The invention also relates to a new protein, characterized by the factthat it comprises or is constituted by:

-   -   sequence SEQ ID NO: 2,    -   or any sequence derived from SEQ ID NO: 2, in particular by        substitution, suppression or addition of one or more amino        acids, having the property of transporting linear polyols in        plants and fungi,

such as polyols having a main chain containing 5 to 8 carbon atoms, inparticular 5 to 7 carbon atoms, in particular 6 carbon atoms, thesepolyols being advantageously chosen from mannitol, sorbitol, dulcitol,galactitol, inositol, ribitol and xylitol, and being in particularmannitol,

-   -   any homologous sequence of SEQ ID NO: 2, preferably having a        homology of at least approximately 50% with sequence SEQ ID NO:        2 and possessing the property of transporting, in plants and        fungi, polyols as defined above,    -   or any fragment of one of the sequences defined above, on the        condition that it has the property of transporting, in plants        and fungi, polyols as defined above, in particular any fragment        being constituted of at least approximately 10 amino acids        adjacent in the sequence SEQ ID NO: 2.

The property of transporting polyols presented by a polyol carrier canbe verified by one or other of the following tests:

-   -   the use of S. cerevisiae yeast or    -   the use of purified plasmic membrane of phloem vesicles.

The use of the yeast Saccharomyces cerevisiae (Noiraud et al., 2000)comprises the transformation of yeasts with the nucleotide sequence tobe tested, these yeasts are capable of growing on said polyol. To verifythat the polyol is transported in these yeasts, radioactively labelledpolyol can be used. For each experiment, a control is perfected with astrain of yeast incapable of growing on the polyol, and which does nottransport said polyol.

The test using a purified plasmic membrane from phloem vesicles is thatdescribed by Salmon et al. (1995).

According to an advantageous embodiment of the invention, the protein ofthe invention, as defined above, is characterized in that it isconstituted by the sequence SEQ ID NO: 2.

The invention also relates to the protein fragments as defined above,chosen from the following sequences:

-   Ala Cys Ala Leu Leu Ala Ser Met Asn Ser Ile Leu Leu Gly Tyr Asp Thr    Gly Val Leu Ser Gly Ala Ser Ile (SEQ ID NO: 11) delimited from the    amino acid in position (26) to the amino acid in position (50) of    the sequence SEQ ID NO: 2,-   Gln Ile Glu Ile Ile Ile Gly Ile Ile Asn Ile Tyr Ser Leu Leu Gly Ser    Ala Ile Ala Gly (SEQ ID NO: 12) delimited from the amino acid in    position (62) to the amino acid in position (82) of the sequence SEQ    ID NO: 2,-   Tyr Thr Met Val Leu Ala Gly Ile Ile Phe Phe Leu Gly Ala Ile Phe Met    Gly Leu Ala (SEQ ID NO: 13) delimited from the amino acid in    position (92) to the amino acid in position (111) of the sequence    SEQ ID NO: 2,-   Phe Leu Met Phe Gly Arg Phe Val Ala Gly Ile Gly Val Gly Tyr Ala Met    Met Ile Ala Pro Val Tyr Thr Ala (SEQ ID NO: 14) delimited from the    amino acid in position (116) to the amino acid in position (140) of    the sequence SEQ ID NO: 2,-   Phe Leu Thr Ser Phe Pro Glu Val Phe Ile Asn Ser Gly Val Leu Leu Gly    Tyr Val Ser Asn Phe Ala Phe Ala (SEQ ID NO: 15) delimited from the    amino acid in position (150) to the amino acid in position (174) of    the sequence SEQ ID NO: 2,-   Ile Met Leu Gly Ile Gly Ala Phe Pro Ser Val Ala Leu Ala Ile Ile Val    Leu Tyr Met (SEQ ID NO: 16) delimited from the amino acid in    position (184) to the amino acid in position (203) of the sequence    SEQ ID NO: 2,-   Ala Ala Ile Thr Gly Ile Gly Ile His Phe Phe Gln Gln Ala Cys Gly Ile    Asp Ala Val Val Leu (SEQ ID NO: 17) delimited from the amino acid in    position (281) to the amino acid in position (302) of the sequence    SEQ ID NO: 2,-   Leu Leu Ala Thr Ile Ala Val Gly Val Cys Lys Thr Val Phe Ile Leu Ile    Ser Thr Phe (SEQ ID NO: 18) delimited from the amino acid in    position (320) to the amino acid in position (339) of the sequence    SEQ ID NO: 2,-   Leu Met Leu Thr Ser Met Gly Gly Met Val Ile Ala Leu Phe Val Leu Ala    Gly Ser Leu Thr Val (SEQ ID NO: 19) delimited from the amino acid in    position (349) to the amino acid in position (370) of the sequence    SEQ ID NO: 2,-   Gly Gly Leu Ala Ile Phe Thr Val Tyr Ala Phe Val Ser Ile Phe Ser Ser    Gly Met Gly Pro Ile Ala Trp Val Tyr (SEQ ID NO: 20) delimited from    the amino acid in position (382) to the amino acid in position (407)    of the sequence SEQ ID NO: 2,-   Cys Ser Ile Gly Val Ala Val Asn Arg Gly Met Ser Gly Ile Ile Gly Met    Thr Phe Ile Ser (SEQ ID NO: 21) delimited from the amino acid in    position (421) to the amino acid in position (441) of the sequence    SEQ ID NO: 2, and-   Ala Phe Leu Leu Phe Ala Val Val Ala Ser Ile Gly Trp Val Phe Met Tyr    Thr Met Phe (SEQ ID NO: 22) delimited from the amino acid in    position (451) to the amino acid in position (470) of the sequence    SEQ ID NO: 2.

The invention also relates to a nucleotide sequence coding for a proteinas defined above.

An advantageous DNA sequence of the invention comprises or isconstituted by:

-   -   the nucleotide sequence SEQ ID NO: 1,    -   or any nucleotide sequence derived by degeneration of the        genetic code, of the sequence SEQ ID NO: 1 coding for a protein        represented by SEQ ID NO: 2,    -   or any nucleotide sequence derived, in particular by        substitution, suppression or addition of one or more        nucleotides, of the sequence SEQ ID NO: 1 coding for a protein        derived from SEQ ID NO: 2, as defined above,    -   or any homologous nucleotide sequence of SEQ ID NO: 1,        preferably having a homology of at least approximately 35% with        the sequence SEQ ID NO: 1 coding for a homologous protein of SEQ        ID NO: 2, as defined above,    -   or any fragment of the nucleotide sequence SEQ ID NO: 1 or of        the nucleotide sequences defined above, said fragment being        preferably constituted of at least approximately 30 nucleotides        adjacent in said sequence,    -   or any complementary nucleotide sequence of the above-mentioned        sequences or fragments,    -   or any nucleotide sequence capable of hybridizing in stringent        conditions with the complementary sequence of one of the        abovementioned sequences or fragments.

By stringent conditions of hybridization is understood:

-   -   temperature of hybridization: 65° C.,    -   hybridization medium: sodium phosphate buffer 250 mM, pH 7.2;        6.6% (w/v) of SDS; 1 mM EDTA; 1% (w/v) of bovine serum albumin,    -   washing temperature: 65° C.,    -   successive rinsing media:        -   2×SSC (1.75% NaCl; 0.88% sodium citrate), SDS 0.1%        -   1×SSC (0.875% NaCl; 0.44% sodium citrate), SDS 0.1%        -   0.5×SSC (0.44% NaCl; 0.22% sodium citrate), SDS 0.1%.

The invention also relates to the fragments of nucleotide sequences asdefined above, chosen from the following sequences:

-   GCT TGT GCT CTT TTA GCT TCC ATG AAT TCC ATC TTA CTC GGC TAT GAC ACC    GGA GTG TTG AGT GGA GCA TCA ATA (SEQ ID NO: 23) delimited from the    nucleotide in position (92) to the nucleotide in position (166) of    the sequence SEQ ID NO: 1,-   CAA ATC GAA ATA ATC ATC GGA ATC ATC AAC ATC TAC TCT CTT CTT GGT TCG    GCC ATA GCC GGA (SEQ ID NO: 24) delimited from the nucleotide in    position (200) to the nucleotide in position (262) of the sequence    SEQ ID NO: 1,-   TAC ACC ATG GTA CTA GCT GGT ATC ATA TTT TTT CTA GGA GCC ATT TTC ATG    GGG CTT GCT (SEQ ID NO: 25) delimited from the nucleotide in    position (290) to the nucleotide in position (349) of the sequence    SEQ ID NO: 1,-   TTT CTC ATG TTT GGT CGC TTT GTT GCT GGA ATT GGT GTC GGT TAT GCC ATG    ATG ATC GCT CCC GTC TAC ACT GCC (SEQ ID NO: 26) delimited from the    nucleotide in position (362) to the nucleotide in position (436) of    the sequence SEQ ID NO: 1,-   TTC CTC ACT TCT TTT CCT GAG GTT TTC ATT AAT TCT GGT GTG TTG CTC GGG    TAT GTA TCC AAC TTT GCA TTT GCC (SEQ ID NO: 27) delimited from the    nucleotide in position (464) to the nucleotide in position (538) of    the sequence SEQ ID NO: 1,-   ATT ATG CTG GGA ATT GGA GCA TTT CCT TCA GTT GCC TTG GCC ATA ATT GTG    TTA TAT ATG (SEQ ID NO: 28) delimited from the nucleotide in    position (566) to the nucleotide in position (625) of the sequence    SEQ ID NO: 1,-   GCT GCA ATT ACG GGT ATT GGT ATT CAT TTC TTC CAA CAG GCT TGT GGT ATT    GAT GCT GTT GTT TTA (SEQ ID NO: 29) delimited from the nucleotide in    position (857) to the nucleotide in position (922) of the sequence    SEQ ID NO: 1,-   CTC CTT GCG ACA ATT GCT GTT GGA GTC TGC AAA ACA GTC TTT ATT CTG ATA    TCA ACG TTT (SEQ ID NO: 30) delimited from the nucleotide in    position (974) to the nucleotide in position (1033) of the sequence    SEQ ID NO: 1,-   CTG ATG CTA ACA AGT ATG GGG GGT ATG GTT ATT GCT CTA TTT GTA CTG GCA    GGC TCA TTG ACG GTT (SEQ ID NO: 31) delimited from the nucleotide in    position (1061) to the nucleotide in position (1126) of the sequence    SEQ ID NO: 1,-   GGT GGT TTG GCA ATA TTT ACA GTG TAT GCT TTT GTG TCG ATA TTT TCA AGT    GGC ATG GGT CCA ATT GCT TGG GTC TAT (SEQ ID NO: 32) delimited from    the nucleotide in position (1160) to the nucleotide in    position (1237) of the sequence SEQ ID NO: 1,-   TGT AGT ATC GGA GTG GCA GTT AAC CGT GGC ATG AGT GGC ATA ATT GGA ATG    ACA TTT ATA TCG (SEQ ID NO: 33) delimited from the nucleotide in    position (1277) to the nucleotide in position (1339) of the sequence    SEQ ID NO: 1,-   GCA TTC CTT TTA TTT GCT GTG GTT GCA TCT ATC GGA TGG GTC TTT ATG TAC    ACA ATG TTC (SEQ ID NO: 34) delimited from the nucleotide in    position (1367) to the nucleotide in position (1426) of the sequence    SEQ ID NO: 1,

The nucleic acid sequence SEQ ID NO: 23 codes for the protein fragmentSEQ ID NO: 11.

The nucleic acid sequence SEQ ID NO: 24 codes for the protein fragmentSEQ ID NO: 12.

The nucleic acid sequence SEQ ID NO: 25 codes for the protein fragmentSEQ ID NO: 13.

The nucleic acid sequence SEQ ID NO: 26 codes for the protein fragmentSEQ ID NO: 14.

The nucleic acid sequence SEQ ID NO: 27 codes for the protein fragmentSEQ ID NO: 15.

The nucleic acid sequence SEQ ID NO: 28 codes for the protein fragmentSEQ ID NO: 16.

The nucleic acid sequence SEQ ID NO: 29 codes for the protein fragmentSEQ ID NO: 17.

The nucleic acid sequence SEQ ID NO: 30 codes for the protein fragmentSEQ ID NO: 18.

The nucleic acid sequence SEQ ID NO: 31 codes for the protein fragmentSEQ ID NO: 19.

The nucleic acid sequence SEQ ID NO: 32 codes for the protein fragmentSEQ ID NO: 20.

The nucleic acid sequence SEQ ID NO: 33 codes for the protein fragmentSEQ ID NO: 21.

The nucleic acid sequence SEQ ID NO: 34 codes for the protein fragmentSEQ ID NO: 22.

The invention also relates to a recombinant vector, in particularplasmid, cosmid, phage or virus DNA, containing a nucleotide sequence asmentioned above.

The invention also relates to a recombinant vector as defined above,containing the elements necessary for expression in a host cell ofpolypeptides coded by the nucleic acids as defined above, inserted intosaid vector.

According to an advantageous embodiment of the invention, therecombinant vector defined above contains in particular a promoterrecognized by the RNA polymerase of the host cell, in particular aninducible promoter and optionally a transcription or terminationsequence, and optionally a signal and/or anchoring sequence.

According to another advantageous embodiment of the invention, therecombinant vector, such as defined above, contains the elements whichallow the expression of a nucleotide sequence, as defined above, as amature protein or fusion protein.

The invention also relates to a host cell, chosen in particular frombacteria, viruses, yeasts, fungi, plants or the cells of mammals, saidhost cell being transformed, in particular using a recombinant vector asdefined above.

According to an advantageous embodiment of the invention, the host cell,as defined above, contains the regulation elements allowing theexpression of the nucleotide sequence as defined above.

The invention also relates to the product of the expression of a nucleicacid expressed by a host cell transformed as defined above.

The invention also relates to an antibody characterized in that it isdirected in a specific manner against a protein of the invention.

The invention is not limited to polyclonal antibodies; the inventionalso relates to any monoclonal antibody produced by any hybridomacapable of being formed according to standard methods starting from, onthe one hand, animal, in particular mouse or rat, spleen cells, thecells of the animal being immunized against the protein of theinvention, and on the other hand cells of a cell line of myeloma, saidhybridoma being capable of being chosen according to the capacity of thecell line to produce monoclonal antibodies recognizing the protein usedbeforehand for the immunization of the animals.

The invention also relates to a nucleotide probe capable of hybridizingwith any one of the nucleic sequences of the invention.

The invention also relates to the antisense oligonucleotides orantisense messenger RNA, derived from the nucleotide sequences asdefined above.

By modification of the expression of the mannitol carrier, usingantisense oligonucleotides, it can then be determined if a reduction ofthe expression of the mannitol carrier has the result of reducingtolerance to saline stress.

The invention also relates to plant cells containing in their genome anucleotide sequence as defined above.

The invention also relates to the transgenic plants, parts of plants,plant seeds or plant propagation material containing cells such asdefined above.

The invention relates in particular to transgenic plants which, in theirnative state, do not contain or express the gene of the mannitolcarrier, in the genome of which said nucleotide sequence is introduced.

The invention relates in particular to the transgenic plants which, intheir native state, contain or express the gene of the mannitol carrier,in the genome of which said nucleotide sequence is introduced.

The invention also relates to a process for the preparation of arecombinant protein as defined above, comprising the following stages:

-   -   Culture in an appropriate medium of a host cell which has been        transformed beforehand by an appropriate vector containing a        nucleic acid of the invention, and    -   Recovery of the protein produced by the abovementioned host cell        transformed from the abovementioned culture medium or from the        host cell.

For example, a process for the preparation of a transgenic celery asdefined above, comprises the following stages:

-   -   inoculation of the celery tissues,    -   coculture of the celery segments and of A. tumefaciens bacteria,    -   elimination of the A. tumefaciens bacteria,    -   regeneration of the transformed celery plants (Nadel et. al.,        1989).

The nucleotide sequences of the invention can be introduced intoplasmids and be combined with regulation elements for expression ineukaryotic cells. These regulation elements are on the one handtranscription promoters and on the other hand transcription terminators.With the nucleotide sequences of the invention contained in theplasmids, the eukaryotic cells can be transformed with the intention ofexpressing a translatable mRNA which makes the synthesis of a polyolcarrier in the cells possible or with the intention of expressing anon-translatable mRNA, which prevents the synthesis of a polyol carrierendogenous in the cells.

The processes of genetic modification of dicotyledons and monocotyledonsare already known (Gasser et al., 1989). For expression in plants, thenucleotide sequences of the invention must be conjugated withtranscription regulation elements. Such elements, called promoters, arealready known (EP 375091).

In addition, coding regions with the termination signals of thetranscription with which they can be correctly transcribed must beprovided. Such elements are also described (Gielen et al., 1989). Theinitiation region of the transcription can be native and/or homologousas well as foreign and/or heterologous to the plant host. If desired,the termination regions are interchangeable amongst themselves. The DNAsequence of the initiation and termination regions of the transcriptioncan be prepared synthetically or obtained naturally, or obtained from amixture of natural or synthetic DNA constituents. To introduce foreigngenes in higher plants, a large number of cloning vectors are availablewhich include a replication signal for E. coli and a marker which allowsselection of the transformed cells.

For the introduction of the nucleotide sequences of the invention into aplant host cell, in addition to transformation using Agrobacteria, thereare many other techniques. These techniques include the fusion ofprotoplasts, the microinjection of DNA and electroporation, as well asballistic methods and viral infection. Starting from the transformedplant material, whole plants can be regenerated in a suitable medium,containing antibiotics or biocides for the selection. The resultingplants can then be tested for the presence of the DNA introduced. Thereis no particular requirement for the plasmids regarding the injectionand the electroporation. Single plasmids can be used such as the pUCderivatives. The presence of a marker gene is necessary for theregeneration of whole plants from such transformed cells. Thetransformed cells develop in the plants in the usual manner (McCormicket al., 1986). These plants can develop normally and be crossed withplants which possess the same transformed genes or different genes. Theresulting hybrids have the corresponding phenotypic properties.

The DNA sequences of the invention can also be introduced into plasmidsand be combined with regulation elements for an expression inprokaryotic cells.

The DNA sequences of the invention can also be introduced into plasmidswhich allow a mutagenesis or a sequence modification by means of arecombination of DNA sequences in prokaryotic or eukaryotic systems.

The transgenic plants of the invention are in particular characterizedby an increase of the capacity to transport a polyol of the inventionand to accumulate it in the organs from which it is extracted. They canbe used to direct the flow of said polyol with the aid of said carriertowards the organs which accumulate little salt, thus facilitatingextraction.

The invention also relates to a process of screening geneticallymodified plants with at least one nucleotide sequence of interest whichcomprises the following stages:

-   -   the transformation of plant cells with a vector containing an        insertion sequence, said insertion sequence comprising the        nucleotide sequence of interest and a nucleotide sequence coding        for a polyol carrier as defined above,    -   the culture of the cells thus transformed on a medium containing        said polyol as an only source of carbon, to obtain transgenic        plants or fragments of transgenic plants containing said        insertion sequence.

This process relates to plants not synthesizing polyol or plants whichsynthesize it.

It concerns, more particularly, the transformation of fragments or ofplant cells with a nucleotide sequence coding for a polyol carrier, inparticular mannitol. The screening is then carried out on a mediumcontaining said polyol as the only source of carbon. The plantsexpressing the polyol carrier thus have an advantage in growth over thenon-transformed plants. At this stage, it can be supposed that any plantis capable of using said polyol as a source of carbon. However, it canprove necessary to do a co-transformation with a gene coding a proteincapable of degrading said polyol. The use of an active promoter only inthe initial phases of regeneration or inducible by a simple compoundmakes it possible to restrict the expression of the polyol carrier tothe selection phases.

The invention therefore concerns a simple selection system based on aplant gene which is no longer necessary once the selection is finishedand on the use of a natural product as a selection agent. This systemavoids having to resort to the use of products likely to be toxic, suchas antibiotics.

The invention also relates to a process for obtaining transgenic plantsresistant to pathogens, which comprises the following stages:

-   -   transformation of plant cells with a nucleotide sequence coding        for a polyol carrier as defined above,    -   culture of the thus transformed cells to obtain transgenic        plants or fragments of transgenic plants.

This process relates to the transformation of plants not synthesizingpolyol or plants which synthesize it, with a nucleotide sequence of apolyol carrier, in particular mannitol, placed either under the controlof a ubiquist promoter (type CaMV 35S) or under the control of aninducible promoter in response to the attack of the pathogen. Theusefulness resides in the fact that the plant, in transporting morepolyol, produced by the pathogen, towards its own cells, suppresses oneof the means of defence put in place by the pathogen to fight againstthe activated oxygen released by the plant in response to this attack.

In order to increase the effectiveness of the process, the expression ofthe polyol carrier can be conjugated with an enzyme degrading saidpolyol.

The invention relates to a process for obtaining transgenic plantsresistant to saline stress, which comprises the following stages:

-   -   transformation of plant cells with a nucleotide sequence coding        for a polyol carrier as defined above,    -   culture of the cells thus transformed to obtain transgenic        plants or fragments of transgenic plants.

This process relates to the transformation of plants not synthesizingpolyol or plants which synthesize it with a nucleotide sequence codingfor a polyol carrier placed under the control of a phloem-specificpromoter (or of the promoter of the polyol carrier). If the plantsynthesizes said polyol, the increase of the transport of said polyolcould lead to an accumulated tolerance to saline stress. In the oppositecase, it is also advisable to introduce genes allowing the synthesis ofsaid polyol, but limiting this synthesis to the leaves in order to avoidharmful effects on the growth of the plant.

DESCRIPTION OF THE FIGURES

FIG. 1 represents the growth test of the yeast MaDH4 expressing theproteic sequence AgMaT1. The cDNA of AgMaT1, under the control of thepromoter ADH1, was introduced in the cells of the MaDH4 strain, and thegrowth of the transformed cells on mannitol was studied. The transformedcells were grown on the SC (synthetic complete) liquid medium withouttryptophan containing either 2% glucose (SC-glu) or 2% mannitol(SC-mann).

MaDH4-YEP112A1XE: MaDH4 containing the empty plasmid;

MaDH4-AgMaT1: MaDH4 containing the plasmid with the nucleic acid ofAgMaT1.

The white squares correspond to MaDH4 yeasts transformed with the emptyplasmid YEP112A1XE (defined hereafter) and grown on SC-glucose medium.

The black squares correspond to MaDH4 yeasts transformed with the emptyplasmid YEP 112A1XE (defined hereafter) and grown on SC-mannitol medium.

The white circles correspond to MaDH4 yeasts transformed withAgMaT1/YEP112A1XE (plasmid YEP112A1XE containing the nucleic acid ofAgMaT1) and grown on SC-glucose medium.

The black circles correspond to MaDH4 yeasts transformed withAgMaT1/YEP112A1XE (plasmid YEP112A1XE containing the nucleic acid ofAgMaT1) and grown on SC-mannitol medium.

The curves represent the evolution according to the absorbance time (at600 nm) of the yeast cultures. This increase of absorbance correspondsin fact to an increase of the number of yeasts in the culture medium andis representative of the growth rate of the yeasts. Therefore the yeaststransformed with the plasmids YEP112A1XE and AgMaT1/YEP112A1XE grow onglucose but only the yeasts transformed with the AgMaT1/YEP112A1XEplasmid are capable of growing on mannitol. It is therefore proof thatAgMaT1 codes for a mannitol carrier.

FIG. 2 represents the absorption of mannitol in cells of S. cerevisiae.The external concentration of mannitol ³H is 500 μM and the pH is 4.5.The squares represent the absorption in transformed cells with thenucleic acid of AgMaT1 whilst the circles represent the absorption incontrol cells transformed with the empty YEP112A1XE plasmid. Only thetransformed cells with the AgMaT1/YEP112A1XE plasmid are capable ofabsorbing the mannitol ³H placed in the external medium.

Material and Methods

Plant Material

Celery plants (Apium graveolens L. dulce variety, Vert d'Elne cultivar)were grown in greenhouses according to the conditions described by Daviset al. (1988). The phloemian bundles were isolated from adult petiolesaccording to the technique described by Daie (1987).

Bacterial Strains and Yeasts.

The following strains were used in this study: Escherichia coli strainsDH5α (supE44, ΔlacU169 (φ80, lacZM15), hsdR17, recA, endA1, gyrA96,thi-1, relA1) (strains commercially available from Clontech). XL1BlueMRF′ (Stratagene) and SOLR (Stratagene) were cultured according tostandard techniques (Sambrook et al., 1989). The Saccharomycescerevisiae MaDH4 strain (ura3, trp1, LEU2, gap1-1, put4-1, uga4-1), thepreparation of which is indicated hereafter, expresses the mannitoldehydrogenase gene of yeast and has been used for the functionalcharacterization of the cDNA of AgMaT1. The 2a strain was obtained bycrossing between the Δα (MATα, ura3, trp1, leu2) (Marcireau et al.,1992) and Σ22574d (MATa, ura3-1, gap1-1, put4-1, uga4-1) (Jauniaux etal., 1987) strains.

Expression Vector in Yeasts

The plasmid YIP 128A1, described in Riesmeier et al. (1992) is used. Themannitol dehydrogenase gene of yeast (YEL070) was amplified by PCR(polymerization chain reaction) using the oligonucleotides MDHPST5(5-GACTCGAGATGACAAAATCAGACGAAACAAC-3) (SEQ ID NO: 35) and MDHBGL3 (5-GAAGATCTTCACACTTGGTCTAAAATTTCC-3) (SEQ ID NO: 36) on the genomic DNA ofthe Saccharomyces Δα strain. The PCR product was cloned in thepBluescript SK vector digested beforehand by Pst1 and BamH1. Aftersequencing to confirm the sequence of the amplified gene, the PCRproduct was digested by Pst1 and Xba1 and cloned in the Pst1/Xba1 sitesof YIP128A1. The construction was integrated into the genome of S.cerevisiae by the EcoV site in the leu2 gene in order to obtain theMaDH4 strain.

5′ RACE-PCR (Rapid Amplification of the cDNA Ends by PCR),

The total RNA of celery leaves was isolated according to the method ofKay et al. (1987). The first cDNA strand was reverse transcribed fromthe total RNA with the degenerated primer(5′-CCNACNCC(G/A)AANGGNA(G/A)NA(G/A)3) (SEQ ID NO: 37) derived from thesequence LLGFGVG (SEQ ID NO: 38) using reverse transcriptaseSuperScript™ II (Stratagene). After degradation of the RNA matrix byRNaseH (Eurogentec), an anchoring primer (dC)₁₆ (SEQ ID NO: 39) wascreated at the 3′ end of the single-stranded cDNA by a deoxynucleotydiltransferase (GibcoBRL). A PCR amplification was carried out using the(dG)₁₆ (SEQ ID NO: 40) and LLGFGVG (SEQ ID NO: 38) primers under thefollowing conditions: 2 minutes at 95° C. then 30 cycles comprisingdenaturation for 2 minutes at 95° C., fixation for 2 minutes at 55° C.and extension for 2 minutes at 72° C. The PCR products were analyzed byagarose gel electrophoresis then cloned in the pGEM-T Easy plasmid(Promega).

Construction and Screening of a cDNA Bank of Celery Phloem

The total RNA of the phloem bundles was isolated according to the methoddescribed by Kay et al. (1987). The polyA+ RNA was purified with thePolyATtract mRNA isolation system (Promega). A unidirectional EcOR1/XhoIbank was constructed in the Uni-ZapXR phage (Stratagene).

The recombinant phages (900,000) were screened with the radioactivelylabelled product of 5′RACE-PCR as probe, in accordance with themanufacturer's protocol (Stratagene). The Hybond TM-N nylon filters(Amersham) were hybridized overnight at 42° C. according to standardconditions (Stratagene). The filters were then rinsed for 15 minutes at42° C. in SSC 2×(SSC 1×=0.15 M NaCl; 0.015 M sodium citrate) with 0.1%SDS, then for 15 minutes in the same medium but at 50° C. and 30 minutesat 50° C. in SSC 1× and 0.1% SDS. The excision in vivo was carried outon the 24 clones which produced a positive signal during the 3successive screening turns. The identified cDNAs were partiallysequenced. The sequence comparisons were carried out on the NationalCenter for Biotechnology Information site. The transmembrane regionswere predicted with the Tmpred program (Hofinann and Stoffel, 1993).

Expression of AgMaT1 in Saccharomyces Cerevisiae

The cDNA of AgMaT1 was ligated in the Pst1-XhoI sites of the yeastvector YEP112A1XE (Riesmeier et al., 1992). This vector allows theexpression of the cDNA under the control of the yeast promoter ADH1. TheMaDH4 yeast cells were rendered competent and transformed according tothe protocol described by Dohmen et al. (1991).

Determination of the Growth Rate

The yeast cultures were grown on SC medium comprising either 2% glucose,or 2% mannitol. Aliquot fractions were taken regularly from the culturesand their absorbance was measured at 600 nm.

Determination of the Mannitol Dehydrogenase Activity

The cells were cultured until in logarithmic growth phase, rinsed indistilled water and resuspended at 80% (weight/volume) in extractionbuffer (50 mM potassium phosphate pH 7.5, 1 mM DTT and 0.5% TritonX100). The cells were broken apart by vortex with glass beads. Thecellular debris was eliminated by centrifuging and the crude extractused for the enzymatic assay. The mannitol dehydrogenase activity wasmeasured at 30° C. according to Quain and Boulton (1987).

Measurement of the Transport of Radiolabelled Mannitol

The cells were cultured until the start of the logarithmic phase(corresponding to an absorbance of 0.6 to 600 nm), washed in distilledwater and resuspended at 1% (weight/volume) in SC medium buffered to pH4.5 with 25 mM MES. A 100 μl aliquot fraction of the cell suspension wasincubated for 60, 120, 180 and 300 seconds in 100 μl of a solutioncontaining of 500 μM [³H]-mannitol. The reaction was stopped by adding 8ml of water at 4° C. and by filtration through glass fibre filters(Sartorius). The radioactivity incorporated in the yeast cells wasdetermined by counting using liquid scintillation (Packard). For theexperiments with inhibitors or competitors, the product was added 30seconds before the radioactive mannitol.

Study of the Expression of AgMaT1 by RT-PCR (Reverse TranscriptionFollowed by Polymerase Chain Amplification)

The total RNA of celery phloem was isolated according to the method ofKay et al. (1987). The first strand of cDNA was reverse transcribed fromthe total RNA with the oligo dT primer by using the reversetranscriptase SuperScript™ II (Stratagene). After degradation of the RNAmatrix by RNaseH (Eurogentec), PCR amplification was carried out usingthe primers 5′ (ATTCTGGTGTGTTGCTCG) (SEQ ID NO: 41) and 3′(CAATGAACAGTATGATGTG) (SEQ ID NO: 42) which allow the amplification of afragment of 661 nucleotides. The PCR conditions were as follows: 2minutes at 95° C. then 30 cycles comprising denaturation for 30 secondsat 95° C., fixation for one minute at 47° C. and extension for 45seconds at 72° C. The PCR products were analyzed by agarose gelelectrophoresis and the intensity of the signal obtained was quantifiedusing Photoshop 5.0 software (Adobe systems Inc.). The extension factorelF4A(10) (Mandel et al., 1995) was used as control gene, the expressionof which is invariable.

Results

Molecular Cloning of AgMaT1

A certain number of proteins which transport sugars or metabolites showsimilarities in their sequences. It has been suggested that thesetransport proteins have evolved from the duplication of an ancestralprotein with 6 transmembrane regions (Maiden et al., 1987). Severalpreserved amino acid regions were identified such as the amino acidsequences at the ends of the 6^(th) and 1^(th) transmembrane domains,PESPR (SEQ ID NO: 43) and PETKG (SEQ ID NO: 44) respectively (Griffithet al., 1992). Comparison between the different glucose carriers (MST1,STP1, STP4, HUP1, HUP3, GLUT1), the D-xylose carrier of L. brevis, thearabinose carrier of E. coli (ARAE), the galactose carrier of E. coli(GALP) and the myo-inositol carriers of yeast (genes ITR1 and ITR2)indicated a preserved region LLGFGVG (SEQ ID NO: 38). This sequence waschosen as matrix for designing the degenerated 5′ RACE primer for PCR.

The first strand of cDNA was reverse transcribed from the entire RNA ofmature celery leaves, primed with a degenerated primer LLGFGVG (SEQ IDNO: 38). After amplification, a band of 1 kb was observed on the agarosegel. All the fragments of this PCR reaction were cloned in a pGEM-T Easyvector (Promega), and several clones were obtained.

In order to obtain an entire clone, a cDNA library was constructedoriginating from phloem bundles isolated from mature celery petioles andthis library was screened with the 5′ RACE-PCR clone. After havingscreened 900,000 transformants, 24 positive clones were identified. Thepositive transformants with inserts of approximately 1.8–2.0 kb werechosen and partially sequenced. One of these clones, called AgMaT1, waschosen for detailed analysis. It contained 1778 pb with an open readingframe which codes for a protein containing 513 amino acids with amolecular mass estimated at 56 kDa. Hydropathic analysis of the deducedsequence of amino acids indicates that AgMaT1 contains 12 transmembranedomains and a long hydrophilic central region of 77 amino acid residues.The amino acid sequence of AgMaT1 was compared with those of thedatabases and it was found that this sequence was related to the sugarcarriers in numerous organisms. The percentage identity of the aminoacids is approximately equal to 50%. However, a greater percentage ofidentity (65%) was found with two optional sugar carriers of Betavulgaris (Beet 1 and Beet 2). An asparagine residue, which is part of anN-glycosylation consensus sequence (Asn372), is situated on the externalside and therefore must be glycosylated. In addition, the consensussequences, which are the common characteristics of the subgroup of sugarcarriers of MFS, are present in AgMaT1. The sequences of PESPRXL (SEQ IDNO: 45) and PETQGRXXXE (SEQ ID NO: 46) were found respectively at theends of the 6^(th) and 12^(th) transmembrane domains, or the(R/K)XGR(R/K) motif between the 2^(nd) and the 3^(rd) and also the8^(th) and 9^(th) transmembrane helices (Griffith et al., 1992).

Note:

The main difficulty encountered during cloning was the total absence ofcharacterisation of such a carrier in any living organism. In fact theonly mannitol carrier is a bacteria mannitol-phospho-transferase (Boeret al., 1994) which carries out both the transport and phosphorylationof mannitol. This combined system is present in bacteria for numeroussubstrates but it does not exist in Eukaryotic organisms. However,according to a first strategy, a first screening of the cDNA bank wascarried out with the part of the gene of mannitol-phospho-transferasecorresponding to the transmembrane field. This screening did not allow aresult to be obtained, which is justified a posteriori by the absence ofsignificant homology between AgMaT1 and themannitol-phospho-transferase.

A second strategy, which turns out not to be operational, is inspired bythat used for identifying the carrier of oligosaccharides in the plants(Patent EP 0,647,273). This consists of complementing the cells ofSaccharomyces cerevisiae with a cDNA bank in an expression vector. Theyeasts are in fact capable of using mannitol as a source of carbon, butthey require a fairly long induction period on mannitol. As has alreadybeen specified, no mannitol carrier has been identified in yeast. Thereasoning being that if a yeast expressed a plant mannitol carrier, thiswould confer on it a growth advantage and that therefore, it would growquicker on a medium containing mannitol. The operation was carried outin this way but none of the cDNAs obtained showed any of thecharacteristics of membrane proteins and in fact resembled transcriptionfactors. The selection system in fact allowed the cDNA which wasinvolved in the expression of yeast genes to be identified and not thecarriers.

Faced with the above difficulties, the Inventors formulated animprobable a priori hypothesis according to which the mannitol carrierwould be part of the super family of glucide carriers described byMarger and Saier (1993). To do this, a species, celery, was used inwhich the existence of a mannitol carrier had been demonstrated (Salmonet al., 1995) and to construct a cDNA bank from the tissue (the phloem)in which the carrier was more expressed. The second stage was theselection of the cDNA obtained according to their capacity to confer thepossibility of transporting mannitol to the yeasts. In these experimentsthe control was the strain of yeast transformed with the emptyexpression plasmid. In this way the mannitol carrier function of thecDNA of AgMaT1 was demonstrated.

During this experiment, other sequences were identified: in total 24clones were obtained. Among all these clones, two were sequenced whichshowed the hydropathy profiles of carriers. The first, M22 (AgMaT1),conferred the ability to transport mannitol to the yeasts whilst thesecond, M7, did not confer it.

Construction of a Strain of Yeast Capable of Metabolizing IntracellularMannitol

Initial studies were carried out in order to characterize the ability ofa yeast to absorb and to metabolize mannitol (Quain and Boulton, 1987).Out of the 40 polyploid strains of S. cerevisiae screened, half of themhave shown good growth on 5% mannitol after long-term adaptation (Quainand Boulton, 1987). As a result, it was decided to test differentstrains of yeasts for their ability to transport and metabolize themannitol and 2 strains were retained. This was firstly carried out byanalyzing the growth characteristics on a medium containing mannitol asthe only carbon source. Σ22574d, generally deficient in a generalcarrier of amino acids and carrier of proline, is incapable of growth ona medium containing mannitol as the only carbon source. On the contrary,Δα was capable of growing on mannitol after long-term adaptation. Afteradaptation, the strain could be maintained successfully on a solidmedium containing 5% mannitol. But maintenance of the adapted Δα strainon a solid medium only containing glucose leads to the total loss of theadapted growth. Such a growth adaptation on mannitol is probably due tothe induction of the key degradation enzymes or the transport permeases.In accordance with the previous observations, NAD⁺ dependant D-mannitoldehydrogenase could be detected in the Δα yeasts (Table 1).

TABLE 1 Activity of mannitol dehydrogenase in different yeast strains.The strains are developed in a liquid medium containing either 2%glucose or 2% mannitol. The results are the averages ± SD of the threeindependent experiments. ND, not detected. Activity (μmol of oxidizedmannitol · (mg of protein)⁻¹ · min⁻¹) Strain glucose mannitol Δα 0.011 ±0.003 0.240 ± 0.007 Σ22574d 0.006 ± 0.002 ND 2a 0.001 ± 0.001 ND MaDH40.410 ± 0.011 ND

In order to obtain an auxotrophy to tryptophan, the Δα strain (Trp⁻) wascrossed with the Σ22574d strain (Trp⁺). Yeast 2a was chosen, whichcannot grow on a medium containing mannitol, with an auxotrophy totryptophan and to leucine. No mannitol dehydrogenase activity wasdetected in cells 2a (Table 1). It was necessary to introduce a limitedmannitol hydrolysis activity inside the yeast. The cDNA of the gene ofthe yeast mannitol dehydrogenase was cloned in YIP128A1 under thecontrol of the ADH1 promoter and it was integrated in a stable manner inthe leu2 gene of 2a. Several transformants have shown a mannitoldehydrogenase activity. The strain with the most significant activity,called MaDH4, was used for the subsequent analyses (Table 1).

Heterologous Expression of the AgMaT1 Protein

For a subsequent characterization of the function of the AgMaT1 protein,it was necessary to express the carrier in a functional manner in aheterologous system such as yeast cells. The cDNA of AgMaT1 wassub-cloned in the PstI/XhoI sites of the YEP 112A1XE shuttle vectorwhich has a promoter/terminator box of the gene of alcohol dehydrogenaseADH1 of S. cerevisiae (Riesmeier et al., 1992). The competent MaDH4cells were transformed with this construction and YEP 112A1XE was usedas control.

All of the constructions were firstly tested for their ability to growon mannitol as the only carbon source. As indicated in FIG. 1, the MaDH4strain, transformed with the empty plasmid YEP 112A1XE is not capable ofgrowing on mannitol. The cells expressing AgMaT1 could grow very well onthis polyol. In order to directly test the ability of the transformedcells to transport mannitol, the yeast cells were incubated in a mediumcontaining [³H]-mannitol for a few seconds to several minutes, the cellswere washed and the radioactivity absorbed was measured by electricscintillation counting. FIG. 2 indicates that the transport of mannitolin the control cells of S. cerevisiae is negligible. However, the MaDH4yeast strains, expressing AgMaT1, transport the [³H]-mannitol at highspeeds when they grow on a medium containing mannitol. The same resultis obtained with the cells of transformed yeast growing on glycerol(data not indicated).

Other polyols such as dulcitol, sorbitol, xylitol, myo-inositol appearcapable of inhibiting by half the absorption of mannitol. The oside formof mannitol, mannose, appears to be recognized by AgMaT1.

Variation of the Expression of AgMaT1 During Saline Stress

The expression of AgMaT1 was monitored in plants having been subjectedto saline stress for 4 weeks (daily watering with 300 mM of NaCl,Noiraud et al., 2000). The phloem of these plants as well as of thecorresponding control plants (watered with water not containing NaCl)was removed in order to extract the RNA which was used to carry outRT-PCR reactions. If the expression of AgMaT1 in the phloem of thecontrol plants is taken as base 100, the expression of AgMaT1 in thephloem of plants treated with NaCl is 500%, which represents a verysignificant stimulation and is in accordance with the role of AgMaT1 insaline stress tolerance in celery.

Transformation Protocol of Petioles or Leaves of Celery

Celery plants (approximately 10 cm in height) regenerated fromembryogenic cells are used as plant material for the transformation.

Inoculation of the Celery Tissues

Agrobacterium tumefaciens bacteria are cultured for 24 hours at 28° C.under agitation in LB medium (Liquid Broth: 1% tryptone, 0.5% autolyticextract of yeast, 0.5% NaCl) with the appropriate antibiotic.

The petioles of celery plants are fragmented into sections ofapproximately 0.5 cm. For each fragment, a longitudinal section isproduced. The celery segments are incubated in MS medium (Murashige &Skoog) 1× (normal concentration, i.e. no dilution) liquid containing1/25^(th) of the culture of Agrobacterium tumefaciens bacteria for 60minutes at ambient temperature.

Composition of the MS Medium

Macro-elements CaCl₂  2.99 mM KH₂PO₄  1.25 mM KNO₃ 18.79 mM MgSO₄  1.50mM NH₄NO₃ 20.61 mM

Vitamins Glycine 26.64 mM Myo-inositol 0.56 mM Nicotinic acid 4.06 μMPyridoxine-HCl 2.43 μM Thiamine-HCl 0.30 μM

Micro-elements CoCl₂, 6 H₂O  0.11 μM CuSO₄, 5 H₂O  0.10 μM FeNaEDTA 0.10 μM H₃Bo₃  0.10 μM KI  5.00 μM MnSO₄, H₂O  0.10 mM Na₂MoO₄, 2 H₂O 1.03 μM ZnSO₄, 7 H₂O 29.91 μMThe excess bacteria are then removed from the celery segments byarranging them on absorbent paper for 2–3 minutes.

Coculture of the Celery Segments and the A. Tumefaciens Bacteria

The cambial surface of the celery segments is left in contact with thegelosed regeneration medium RM. The Petri dishes are placed in a chamberair-conditioned at 25° C. for 48 hours and subjected to light/darkcycles of 16 hours/8 hours.

Elimination of the A. Tumefaciens Bacteria

After coculture for 48 hours, the celery segments are removed form thedishes of RM medium and transferred into MS 1 × liquid supplemented withcefotaxime at a final concentration of 250 μg/mL. After incubation for60 minutes, the celery segments are dried on absorbent paper for 2–3minutes.

Regeneration of Transformed Celery Plants

The cambial surface of the celery segments is left in contact with agelosed callogenesis initiation medium CIM. The CIM Petri dishes areplaced in a chamber air-conditioned at 25° C. and subjected tolight/dark cycles of 16 hours/8 hours until the development of calluses(2–3 weeks). The celery segments are then transferred onto a gelosedorganogenesis induction medium OIM (2–3 weeks). After the appearance ofbuds, these are removed and placed on gelosed rooting medium RM. A fewweeks (3–4 weeks) are necessary for the development of young celeryshoots.

Composition of the Media

Regeneration Medium RM

MS 1× Mannitol 3.0% Saccharose 1.5% Casein hydrolysate 100.0 mg/L6-Benzylaminopurine (BAP)  1.0 mg/L α-naphthylacetic acid (NAA)  0.1mg/L Gibberellic acid (GA₃)  0.1 mg/L Agar 0.8%

Callogenesis Initiation Medium (CIM)

MS 1× Mannitol 3.0% Saccharose 1.5% Casein hydrolysate 100.0 mg/L6-Benzylaminopurine (BAP)  1.0 mg/L α-naphthylacetic acid (NAA)  0.1mg/L Gibberellic acid (GA₃)  0.1 mg/L Kanamycin 125.0 mg/L Cefotaxime200.0 mg/L Agar 0.8%

Organogenesis Induction Medium (OIM)

MS 1× Mannitol 3.0% Saccharose 1.5% Casein hydrolysate 100.0 mg/L6-Benzylaminopurine (BAP)  1.0 mg/L Gibberellic acid (GA₃)  0.1 mg/LKanamycin  75.0 mg/L Cefotaxime 200.0 mg/L Agar 0.8%

Rooting Medium (RM)

MS 1× Mannitol 3.0% Saccharose 1.5% Casein hydrolysate 100.0 mg/Lα-indolyacetic acid (IAA)  0.1 mg/L Kanamycin  75.0 mg/L Cefotaxime200.0 mg/L Agar 0.8%

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1. A protein, comprising: sequence SEQ ID NO:
 2. 2. An isolatedpolypeptide according to claim 1, wherein said polypeptide consists ofSEQ ID NO: 2.